10966
Biochemistry 2005, 44, 10966-10976
Crystal Structure of the D94S/G98E Variant of Rat R-Parvalbumin. An Explanation for the Reduced Divalent Ion Affinity† John J. Tanner,‡,§ Sayeh Agah,§,| Yong-Hwan Lee,‡,⊥ and Michael T. Henzl*,§ Department of Chemistry, UniVersity of Missouri, Columbia, Missouri 65211, and Department of Biochemistry, UniVersity of Missouri, Columbia, Missouri 65211 ReceiVed April 27, 2005
ABSTRACT: Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat R-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca2+ affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the R D94S/G98E variant. The Ca2+ coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the R 94/98E variant in both Na+- and K+-containing buffers. Although the Ca2+ and Mg2+ affinities are higher in K+ solution, the increases are comparable to those observed for wild-type R. Consistent with that finding, the apparent Na+ stoichiometry, estimated from stability studies conducted as a function of Na+ concentration, is 1.0 ( 0.1, identical to that of wild-type R. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the Cγ atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.
Although Ca2+ fulfills several vital extracellular functions, notably in bone formation and blood coagulation, contemporary Ca2+ research is focused largely on its intracellular role in signal transduction. Supplied with an appropriate stimulus, virtually any eukaryotic cell will exhibit transient increases in cytosolic Ca2+ concentration, from a restingstate level between 20 and 100 nM to a maximum approaching 1 µM (1, 2). These Ca2+ signals regulate myriad cellular processes, achieving specificity through spatial and temporal variations. Ca2+ exerts its influence through interactions with a multitude of Ca2+-binding proteins. Many of these belong to the EF-hand protein family, named for its characteristic metal ion-binding motif (3-5). Certain EF-hand proteins, notably, calmodulin and troponin C, play overt regulatory roles. Others, notably calbindin and parvalbumin, serve as cytosolic Ca2+ buffers, modulating the amplitude and duration of the Ca2+ signals. The 30-residue EF-hand motif consists of a 12-residue ionbinding loop flanked by short helical segments. Within the central loop, the liganding moieties are arrayed in pseudo†
This work was supported by NSF Grant MCB0131166 (to M.T.H. and J.J.T.). * To whom correspondence should be addressed. Telephone: (573) 882-7485. Fax: (573) 884-4812. E-mail:
[email protected]. ‡ Department of Chemistry. § Department of Biochemistry. | Present address: Department of Orthopedic Surgery, University of California, Los Angeles, CA 90095-6902. ⊥ Present address: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803.
octahedral fashion around the bound ion and, accordingly, are indexed by a Cartesian coordinate system (+x, +y, +z, -y, -x, and -z). The -y position is occupied by a mainchain carbonyl; the -x ligand is frequently a water molecule, and the -z ligand is a nearly invariant glutamyl residue. The remaining ligands are side-chain oxygen atoms (carboxylate, carbonyl, or hydroxyl). The coordination sphere typically contains three or four carboxylates. Because the -z glutamate binds Ca2+ in a bidentate manner, the coordination is actually pentagonal bipyramidal. The +y, +z, -y, and -z ligands lie in the equatorial plane, and the +x and -x species contribute the axial ligands. Despite their general overall similarity, EF-hand motifs exhibit a broad range of divalent ion affinities. It is increasingly evident that the metal ion binding properties of a particular EF-hand site are influenced by local- and longrange structural features. For example, binding site I in calmodulin and the EF site1 of parvalbumin have identical inner coordination spheres, consisting of three aspartyl 1 Abbreviations: CD, circular dichroism; CD site, parvalbumin metal ion-binding site flanked by the C and D helical segments; DSC, differential scanning calorimetry; EDTA, ethylenediaminetetraacetic acid; EF site, parvalbumin metal ion-binding site flanked by the E and F helical segments; EGTA, ethylene glycol bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ITC, isothermal titration calorimetry; LB, Luria-Bertani; NMR, nuclear magnetic resonance; Pi, phosphate; PV, parvalbumin; 94/98, parvalbumin variant in which Asp-94 has been replaced with serine and Gly-98 has been replaced with either aspartate (94/98D) or glutamate (94/98E).
10.1021/bi050770t CCC: $30.25 © 2005 American Chemical Society Published on Web 07/26/2005
Structure of R D94S/G98E carboxylates (+x, +y, +z), a backbone carbonyl (-y), a water molecule (-x), and the bidentate glutamyl carboxylate (-z). Nevertheless, their apparent Ca2+ association constants differ by nearly 4 orders of magnitude. The basis for this disparity is now reasonably well understood. Specifically, the binding of Ca2+ to calmodulin, but not parvalbumin, provokes an energetically costly solvent exposure of apolar surface area. A detailed appreciation of integrated Ca2+ signaling activity requires a comprehensive understanding of the kinetics and thermodynamics of the relevant Ca2+ pumps, channels, and binding proteins. For nearly three decades, investigators have sought to identify the physical basis for variations in EF-hand divalent ion affinity. This effort is complicated by the profound influence of higher-order structural considerations. Our work in this area has focused on structure-affinity relationships in the parvalbumin family. Parvalbumins are small (Mr ) 12 000) vertebrate-specific proteins believed to act as cytosolic Ca2+ buffers (6, 7). They contain two Ca2+-binding sites, the CD and EF sites, named for the helical segments flanking the ion-binding loops. In fact, the “EF-hand” motif is named after the EF site in carp parvalbumin, where it was first observed (8). The parvalbumin (PV) family includes two sublineages, R and β, that can be distinguished on the basis of isoelectric point, C-terminal helix length, and lineage-specific sequence eccentricities (9, 10). Mammals express one isoform from each lineage (11). Although the sequences of the two proteins from rat are 49% identical (12, 13), they exhibit disparate divalent ion binding properties. In 0.15 M KCl and 0.025 M HepesKOH (pH 7.4), the binding of Ca2+ is 5.5 kcal/mol more favorable for R (14). As part of an inquiry into the basis for this disparate behavior, we produced and characterized a series of “siteinterconversion” variants in both the rat R and β backgrounds (15-17). The ligand arrays in the parvalbumin CD and EF sites differ at the +z and -x positions: serine and glutamate (aspartate in β) in the CD site and aspartate and glycine in the EF site, respectively. The combined S55D and E59G (D59G in β) mutations produce an EF-like array in the confines of the CD site. Conversely, the D94S and G98E (G98D in β) mutations produce a CD-like site in the confines of the EF site. The impact of these mutations has been discussed at length elsewhere (16, 17). Significantly, the 94/ 98 mutations markedly decrease the EF-site Ca2+ affinity, regardless of whether they are made in R or β or whether Gly-98 is replaced with aspartate or glutamate. Because the basis for this effect was unclear, we have obtained highresolution structural data on the R D94S/G98E variant and extended our previous metal ion binding studies. The results of this work offer insight into the divalent ion binding behavior of the 94/98 variants. MATERIALS AND METHODS Protein Purification. R 94/98 was isolated by a minor modification of the procedure described previously for wildtype R (18, 19). Protein concentrations were determined spectrophotometrically, employing an extinction coefficient at 258 nm of 1600 M-1 cm-1. Residual divalent metal ions were removed from protein solutions and buffers when they were passed over a column of EDTA-agarose (20), prepared
Biochemistry, Vol. 44, No. 33, 2005 10967 Table 1: Data Collection and Refinement Statisticsa PDB entry space group unit cell parameters no. of crystals no. of protein molecules in the asymmetric unit diffraction resolution (Å) no. of observations no. of unique reflections completeness (%) mean I/σI Rmerge no. of protein atoms no. of calcium ions no. of water molecules Rcryst Rfreeb rmsdc bond lengths (Å) bond angles (deg) Ramachandran plotd favored (%) allowed (%) generously allowed (%) disallowed (%) average B-factors (Å2) protein calcium ions solvent
1XVJ P21 a ) 30.7 Å, b ) 57.0 Å, c ) 56.5 Å, β ) 105.8° 1 2 25-1.8 (1.85-1.80) 50844 16037 92 (90) 25.3 (9.6) 0.066 (0.224) 1653 4 199 0.197 (0.261) 0.236 (0.348) 0.010 1.2 93.9 6.1 0.0 0.0 17 15 22
a Values for the outer resolution shell of data are given in parentheses. The 5% Rfree test set. c Compared to the Engh and Huber force field (32). d The Ramachandran plot was generated with PROCHECK (35).
b
as described elsewhere (21). Protein preparations thus treated contained
